tbuckley/cabot-search huggingface dataset:
P445: A SYSTEMS-BASED APPROACH TO UNVEIL DRUG RESISTANCE IN FLT3-ITD AML
Authors: Francesca Sacco, Giorgia Massacci, Venafra Veronica, Valeria Bica, Sara Latini, Thomas Fischer, Dimitrios Mougiakakos, Martin Böttcher, Livia Perfetto, Natalie Krahmer, Felix Klingelhuber, Monia Pugliese
Journal: HemaSphere (2023)

Background: Internal tandem duplications (ITDs) in FLT3 gene occurs in about 30% of patients with a de novo AML diagnosis and are generally associated with poor prognostic outcomes. The different FLT3-ITD insertion site impacts the sensitivity to tyrosine kinase inhibitors (TKIs) treatment, influencing patients’ clinical outcome. Specifically, the ITD insertion in the tyrosine kinase domain (TKD) is associated with chemoresistance, relapse and lower overall survival. Aims: Our work aims to elucidate the molecular mechanisms driving the different sensitivity of FLT3-ITD cells to tyrosine kinase inhibitors (TKIs). Additionally, we aim to investigate whether the different prognostic impact of ITD-mutations is due to a diverse cell responsiveness to one of the most commonly prescribed chemotherapeutic drugs, cytarabine. Our final goal is the identification of novel promising therapeutic targets that may improve the prognostic outcome of patients carrying the ITD in the TKD region Methods: Here we tested the hypothesis that the different sensitivity of FLT3-ITD cells to chemotherapeutic drugs may be caused by an extensive rewiring of cell signaling network. Thus, to obtain a comprehensive and unbiased picture of the FLT3-ITD signaling networks, we combined RNA sequencing and mass spectrometry (MS)-based (phospho)proteomics with a newly developed computational strategy, called “SignalingProfiler”. In vitro and in ex vivo assays were performed to in depth characterize the TKI and cytarabine-dependent signaling pathways in FLT3-ITD cells. Results: Our strategy revealed that the different ITD insertion site differently affects cell cycle progression, impairing the activation of crucial kinases. Specifically, we found that the WEE1-CDK1 axis plays a crucial role in TKI therapy failure in patients carrying the ITD in the TKD region. Remarkably, pharmacological inhibition of WEE1 completely rescued the ability of patient-derived primary blasts, carrying the ITD-TKD mutation, to undergo apoptosis in response to midostaurin treatment. Summary/Conclusion Finally, this study demonstrates the impact of ITD-location on cytarabine and TKI sensitivity, identifying clinically relevant effective drug combinations to increase the responsiveness of FLT3-ITD blasts. Additionally, we propose a novel strategy that can be applied to the study of drug resistance and mechanism of action, and can lead to the identification of novel therapeutic targets for combination therapy.Keywords: Proteomics, Signaling, Flt3-ITD, AML
P601: GENETIC DRIVERS IN THE NATURAL HISTORY OF CHRONIC LYMPHOCYTIC LEUKEMIA DEVELOPMENT AS EARLY AS 16 YEARS BEFORE DIAGNOSIS
Authors: Pieter Martijn Kolijn, Florentin Späth, Mouhamad Khouja, Paul Hengeveld, Lina van der Straten, Nikos Darzentas, Magnus Hultdin, James D. Mckay, Christiane Pott, Roel C.H. Vermeulen, Anton Langerak
Journal: HemaSphere (2023)

Background: Chronic lymphocytic leukemia (CLL) is preceded by monoclonal B-cell lymphocytosis (MBL), a CLL precursor state with a prevalence of up to 17% in aged individuals. Previously, we described significant B-cell receptor immunoglobulin heavy chain (BcR IGH) gene repertoire skewing and clonotypic evolution regardless of IGHV mutational status or stereotypy up to 22 years before CLL diagnosis. However, pathobiological drivers during the earliest stages of MBL development remain incompletely characterized.Aims: Here, we aim to gain further insight into the chronological order of key driver mutations during early CLL development through in-depth study of longitudinal blood samples drawn up to 16 years prior to diagnosis. Methods: In this study, we utilized the EuroClonality-NDC panel to sequence recurrently mutated genes in CLL in 39 peripheral blood samples from 16 CLL patients taken up to 16 years prior to diagnosis. Annotation was performed through the EuroClonality-NGS–developed ARResT/Interrogate software tool. We identified somatic variants present (VAF >3%) at the moment of CLL diagnosis and traced these variants in earlier samples to study at what stage in CLL development they had first arisen. For those individuals without a diagnostic sample available, the prediagnostic sample drawn closest to diagnosis was used as a reference instead. Results: Of the 16 CLL patients, 8 (50%) presented with variants of interest in CLL-associated genes such as NOTCH1, ATM, SF3B1 and BRAF. CLL patients with variants of interest included 4 U-CLL cases, 4 M-CLL cases and encompassed several stereotypic CLL subsets (IGLV3-21R110, #2, #7 and #8). ATM variants and the IGLV3-21 R110 mutation were present from the early stages of (pre)MBL development, while NOTCH1, SF3B1, XPO1 and BRAF variants arose closer to diagnosis. We additionally describe variants in FAT1 and PLCG2 as early as 10 years prior to CLL diagnosis. Summary/Conclusion: Overall, our data indicate specific genetic drivers of CLL are associated with early and late stages of CLL development. Keywords: ATM, IgH rearrangment, Notch, B cell chronic lymphocytic leukemia
P604: CHRONIC LYMPHOCYTIC LEUKEMIA CELLS ARE SENSITIVE TO THE APOLIPOPROTEIN E-MEDIATED DISRUPTION OF METABOLIC HOMEOSTASIS
Authors: Federica Nardi, Rosita Del Prete, Roberta Drago, Anthea DI Rita, Laura Pezzella, Sara Ciofini, Laura Tinti, Vittoria Cicaloni, Laura Salvini, Danilo Licastro, Aidan Pezacki, Christopher Chang, Silvia Deaglio, Tiziana Vaisitti, Giuseppe Marotta, Alessandro Gozzetti, Monica Bocchia, Anna Kabanova
Journal: HemaSphere (2023)

Background: Unraveling intrinsic vulnerabilities in chronic lymphocytic leukemia (CLL) represents a key approach to understand molecular basis for its indolence and a path towards designing tailored therapeutic approaches. We previously described that CLL cells are characterized by an ectopic expression of the immunomodulatory cell surface receptor ILT3/LILRB4 (Zurli et al. Blood 2017). Despite functional implications of ILT3 expression have not been fully elucidated, its selective presence on CLL B cells, but not healthy B cells, made us hypothesize that it might influence CLL biology in a considerable way. Recently, a new physiological ligand for ILT3 has been described as the apolipotrotein E (ApoE), an abundant serum protein regulating systemic lipid homeostasis. Aims: Encouraged by the evidence that ApoE is a natural ligand for ILT3, here we investigated the effect of ApoE on CLL cell fitness and the underlying mechanisms of its action. Methods: We expressed lipoprotein-associated recombinant ApoE and used it for in-depth biochemical, transcriptomics and functional in vitro investigation of primary CLL cells from CLL patients and the Richter syndrome xenoografts. Results: We find that, at physiological concentrations, ApoE inhibits CD40-driven proliferation of CLL cells by interfering with two intrinsic CLL cell vulnerabilities. First, we establish that ApoE is a copper-binding protein that alters metal homeostasis of CLL cells and promotes a copper-mediated cell death. Second, ApoE strongly downregulates D-3-phosphoglycerate dehydrogenase (PHGDH), the activity of which is essential to support CLL proliferation. Escape from the ApoE-mediated inhibition is possible in the transformed CLL cells from patients with Richter syndrome. Our fundings made us hypothesize that ApoE could exert its inhibitory effect upon CLL cells in proliferating centers in the tumor niche. Single-cell data suggest that ApoE could be expressed in the CLL microenvironment, and in fact we detected ApoE in the secretome of primary stromal cell cultures derived from the CLL bone marrow. Summary/Conclusion: Our findings suggest a natural suppression mechanism in CLL mediated by ApoE. This protein, serum-derived and locally produced ApoE, can inhibit tumour cell growth by disrupting CLL cells metabolic homeostasis. This vulnerability might be associated with intrinsic hypersensitivity of CLL cells to oxidative stress and their dependence on metabolic rewiring for cellular proliferation. Keywords: B cell chronic lymphocytic leukemia
P701: SYSTEMATIC ANALYSIS OF T LYMPHOCYTES IN MYELOID NEOPLASMS
Authors: Athanasios Tasis, Maria Grigoriou, Kyriaki Katsiki, Anastasia Filia, Nikolaos Paschalidis, Eleftheria Lamprianidou, Apostolos Vasileiou, Konstantinos Liapis, Maria Angelidou, Ioannis Kotsianidis, Ioannis Mitroulis
Journal: HemaSphere (2023)

Background: Myelodysplastic syndrome (MDS) is a diverse group of clonal hematopoietic progenitor cell malignancies, with an elevated risk for progression to acute myeloid leukemia (AML). Although azacitidine is the treatment of choice for patients with MDS and AML, its exact mechanism of action still remains elusive. To date, many unanswered questions remain about the role of adaptive immunity in the disease progression and patients’ response to azacitidine. Aims: Herein, we sought to investigate the immune landscape, of bone marrow samples from patients with low-risk MDS, chronic myelomonocytic leukemia (CMML), high-risk MDS (RAEB-II), and AML. Methods: To this goal, mass cytometry (CyTOF) and flow cytometry were performed on isolated bone marrow mononuclear cells (BMMCs), while scRNA-sequencing was utilized to define the transcriptome profile of bone marrow-derived CD8+ T cells. All samples were obtained before initiation of treatment with azacitidine. Results: The immunophenotypic analysis demonstrated differences in T cell populations, which distinguishes MDS patients from those with AML. In particular, AML patients showcased an increased expression of the chemokine receptor CXCR3 on cytotoxic CD8+ T-lymphocytes and a reduced percentage of naive CD4+ T-lymphocytes. Most importantly, a CD8+ T-lymphocyte subpopulation (CXCR3+CD57+) was identified, which differentiates azacitidine responders (Complete Remission; CR) from non-responders (Failure; FAIL) (Figure 1). Moreover, unbiased clustering of our scRNA-seq data, partitioned the CD8+ T cells into 11 homogenous cell populations (clusters) and identified molecular pathways that distinguish AML from MDS. Additionally, the proportion of cells forming the cluster with the most enhanced cytotoxic signature was significantly elevated in patients with AML compared to MDS. Summary/Conclusion: The present study highlights the heterogeneity of T cells in myeloid neoplasms, while introducing a new CD8+ subpopulation (CXCR3+CD57+) as a potential biomarker of response in AML and MDS. Acknowledgment: This research has been co‐financed by the European Regional Development Fund of the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH – CREATE – INNOVATE (project code: T2EDK-02288, MDS-TARGET).Keywords: MDS, RNA-seq, Flow cytometry, AML
P607: INTEGRATING MULTI-OMICS TO REVEAL THE CLONAL EVOLUTIONARY CHARACTERISTICS IN CLL PATIENTS WITH ZANUBRUTINIB RESISTANCE
Authors: Huayuan Zhu, Yeqin Sha, Yi Miao, Shuchao Qin, Luomengjia Dai, Tonglu Qiu, Wei Wu, Yi Xia, Lei Fan, Wei Xu, Jianyong LI
Journal: HemaSphere (2023)

Background: The drug-resistant mechanisms of the first-generation Bruton’s tyrosine kinase (BTK) inhibitor, ibrutinib, has been extensively explored in Chronic Lymphocytic Leukemia (CLL) patients. However, the resistant mechanisms of the second-generation BTK inhibitor such as zanubrutinib remained largely unexplored. Aims: To assess the clonal characteristics in CLL patients with zanubrutinib resistance. Methods: We retrospectively identified 8 CLL patients with zanubrutinib resistance. Deep targeted-gene NGS and ddPCR detecting BTK and PLCG2 mutation were assessed in serial samples. scRNA-seq of matching peripheral blood (PB) and lymph node (LN) were performed in 3 zanubrutinib-resistant patients showing progressive lymphadenopathy. Results: Median time from zanubrutinib initiation to progression was 30.5 months, including 2 Richter Transformation (RT) patients after 13 months’ treatment. NGS showed that BTK Cys481 mutation was detected in 5 of 8 patients (1 patients also harbored BTK Leu528Trp with low frequency) and PLCG2 mutation in one patient without BTK Cys481 mutation. For 3 patients with undetectable BTK mutation by NGS, ddPCR were performed and BTK Cys481Ser was identified in one patient, suggesting spatial heterogeneity. Clonal evolution model were depicted in patients available for longitudinal NGS. TP53, EGR2, NOTCH1 and SF3B1 were main driving clones during zanubrutinib resistance. scRNA-seq analysis showed that RT showed significantly higher expression of MCL-1 while progressive CLL showed relatively higher expression of BCL-2 (p<0.0001), indicating the shift of BCL-2 family dependence among different disease characteristics. Summary/Conclusion: NGS and ddPCR should be used comprehensively to evaluate the emergence of resistant clones. BTK Cys481 and Leu528 were two main BTK mutations leading to zanubrutinib resistance. The shift of BCL2 anti-apoptotic family gene expression indicates that higher MCL-1 expression of RT might lead to insensitivity to venetoclax treatment following zanubrutinib resistance.Keywords: Drug resistance, Bruton’s tyrosine kinase inhibitor (BTKi), B cell chronic lymphocytic leukemia
P467: LONG NON-CODING RNA SNHG29 IS A NOVEL MYELOID LEUKEMIA DEPENDENCY
Authors: Robert Winkler, Dirk Heckl, Michelle Ng, Reinier Boon, Hasan Issa, Raj Bhayadia, Jan-Henning Klusmann
Journal: HemaSphere (2023)

Background: Long non-coding RNAs (lncRNAs) have emerged as developmental stage- and cell type-specific regulators of gene expression. They have been implicated as important regulators of normal and malignant hematopoiesis with untapped therapeutic potential. However, the vast majority of lncRNAs lack comprehensive functional and mechanistic characterization. Aims: In this study, we aimed to identify and characterize lncRNA that contribute to acute myeloid leukemia (AML) and could serve as novel therapeutic targets. Methods: We used a high-throughput CRISPR interference (CRISPRi) approach to screen a signature of lncRNAs overexpressed in pediatric AML patient samples and hematopoietic stem and progenitor cells (HSPCs). We leveraged RNA-targeting CRISPR-class system CasRx and small hairpin RNA (shRNA) to functionally characterize candidate lncRNAs in cell lines and patient material. We assayed proliferation using flow cytometry based competition assays and tested colony-forming capability. Results: To investigate lncRNAs in normal and malignant hematopoiesis, we profiled lncRNA expression in hematopoietic stem and progenitor cells (HSPCs) and pediatric AML blasts from different cytogenetic subgroups and discovered a signature of lncRNAs overexpressed in AML and HSPC. We performed targeted CRISPRi proliferation screens of HSPC/AML lncRNAs (619 genes, 2009 sgRNAs) in a panel of seven AML cell lines. SNHG29, a highly abundant lncRNA transcript on chromosome 17, was found to be selectively required for the proliferation of M07e, a cell line of megakaryoblastic AML (AMKL). In AML patient cohorts, SNHG29 is co-expressed with programs relating to translation, cell cycle and DNA replication. To validate these findings, we targeted SNHG29 using multiple perturbation approaches in cell lines as well as PDX. Both shRNA and CasRx-mediated knockdown of SNHG29 reduced the growth of M07e cells, indicating a transcript-dependent mechanism. Knockdown of SNHG29 in AMKL PDX reduced colony formation in vitro, suggesting the clinical relevance of this lncRNA. Integrating RNA pulldown and transcriptomics data elucidated that SNHG29 is involved in a complex regulatory network regulating hematopoiesis. Summary/Conclusion: Here, we systematically interrogated lncRNAs in AML using CRISPRi screening and identified SNHG29 as a selective myeloid leukemia dependency and potential novel therapeutic target. The findings highlight the power of this approach and offer new avenues for therapeutic intervention in AML. Keywords: Screening, Acute myeloid leukemia
P440: ENHANCER HIJACKING IN COMPLEX-KARYOTYPE AML
Authors: Etienne Sollier, Anna Riedel, Katherine Kelly, Dieter Weichenhan, Ekaterina Jahn, Konstanze Döhner, Hartmut Döhner, Aurélie Ernst, Pavlo Lutsik, Christoph Plass
Journal: HemaSphere (2023)

Background: The total number of structural rearrangements in acute myeloid leukemkia (AML) is generally much lower than in other cancer types. However, AML with a complex karyotype (ckAML) is a rare AML subtype (10-15% of all cases) characterized by the presence of at least three cytogenetic alterations and has a dismal prognosis. It is still poorly understood. Many commonly deleted regions have been identified, for example in 5q, 7q, 12p and 17p, but it is still unclear how they may drive leukemia. In parallel, it has been discovered in the last decade that structural rearrangements can lead to aberrant expression of genes, for example of EVI1 in inv(3) or t(3;3), but no systematic search for such events has been undertaken. Since ckAML cases have many structural rearrangements, it is likely that some of them could lead to enhancer hijacking. Aims: We systematically searched for genes, which can be activated by enhancer hijacking in ckAML. Methods: We performed whole genome sequencing (WGS) and RNAseq of 42 ckAML cases. For each gene, we identified samples which had breakpoints located in the same topologically-associating domain, using the WGS. Then, we tested if the samples with breakpoints in the vicinity of the gene had an outlier high expression for this gene. Enhancer hijacking should lead to the expression of only the rearranged allele, so we filtered for genes with monoallelic expression, based on SNPs detected in WGS and RNAseq. Results: We detected many putative enhancer hijacking events in our cohort of 42 ckAML cases. Among the top 20 candidates were three genes, which have already been reported in the literature as being activated by enhancer hijacking in AML: EVI1, BCL11B and MNX1. This supports the reliability of our method. In addition, we detected several novel interesting candidates and we could for some of them find recurrence of these events in other cohorts. Several enhancers were hijacked, including strong hematopoietic enhancers like MYC, GATA2 or CDK6. Summary/Conclusion: We performed for the first time an exhaustive search for enhancer hijacking events in ckAML, and found both known and novel genes. This provides a better understanding of this deadly and poorly understood disease. Since these events are rare, sequencing even larger cohorts could lead to the identification of even more genes and could help estimate the frequency of these events.Keywords: AML, EVI1, TP53, Complex aberrant karyotype
P657: PATHOGENESIS OF TYROSINE KINASE INHIBITOR–ASSOCIATED VASCULAR TOXICITY IN CHRONIC MYELOID LEUKEMIA
Authors: Ayala Gover Proaktor, Galit Granot, Oren Pasvolsky, Pia Raanani, Avi Leader
Journal: HemaSphere (2023)

Background: Evidence has emerged demonstrating an association between treatment with certain tyrosine kinase inhibitors (TKIs) and vascular adverse events (VAEs) in chronic myeloid leukemia (CML). An increase in VAEs has been documented with ponatinib, nilotinib and dasatinib, but not with imatinib and bosutinib. TKIs are key components in CML treatment, and therefore their mechanism of toxicity is of great interest. Aims: The aim of this study is to understand the molecular mechanisms underlying the vascular toxicity seen in CML patients treated with some of the TKIs. Methods: We used an in-vitro endothelial cell (HUVEC) model to characterize the effects of TKIs on endothelial function via tube formation assay and on endothelial cell gene expression via RNA-seq. An in-vivo hindlimb ischemia mouse model was used to analyze the effect of TKIs on angiogenesis. In both models, clinically relevant doses of TKIs were used. Results: In a HUVEC tube formation assay we show that dasatinib and ponatinib significantly abrogated HUVEC tube formation as apparent by reduction in tube number, junction number and segment length. Imatinib, nilotinib and bosutinib did not affect HUVEC tube formation. The impact of the different TKIs on HUVEC gene expression was assessed via RNA-seq. A clear distinct molecular pattern of expression in nilotinib, ponatinib, and dasatinib treated cells compared to control, imatinib and bosutinib treated cells which were clustered together. Nilotinib, ponatinib, and dasatinib treatments result in a unique HUVEC gene expression profile with differential expression of angiogenesis-related genes. In our in-vivo model, femoral artery was excised to induce ischemia and stimulate angiogenesis. Mice were treated with clinically relevant doses of TKIs and blood perfusion was measured via Doppler imager. In agreement with our in-vitro results, dasatinib treated mice showed a significantly delayed recovery from ischemia and a significant increase in toe necrosis. Imatinib and bosutinib treated mice showed a similar recovery pattern to control mice, corresponding to the in-vitro results. The recovery pattern of ponatinib-treated mice was also similar to control mice, differing from the anti-angiogenic effect that was observed in-vitro. Summary/Conclusion: The in-vivo and in-vitro results of dasatinib treatment are in line with its clinical VAE profile, implicating endothelial toxicity related to dasatinib and supporting a direct effect of dasatinib on vascular pathogenesis. Understanding TKI-induced VAEs pathogenesis could contribute to future development of safer next-generation TKIs and could potentially influence TKI-personalized tailoring for CML patients. Keywords: Angiogenesis, Chronic myeloid leukemia, Tyrosine kinase inhibitor